This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cytochrome c is a model system in protein folding studies and it serves as a testing ground for new methods. Since the kinetic version of 2D-FT, which will be our major tool in this study, is under development at ACERT, we are applying other methods that can report on protein structural rearrangement. They include DQC and double electron-electron resonance (DEER). Dr. Scholes has succeeded in the preparation of iso-Cytochrome c double cysteine mutants and their spin-labeling with nitroxides. He is developing a suitable freeze-quench apparatus using nanofabrication technology at the Cornell Nanofabrication Facility. The micromixer/freeze-quench assembly was designed to capture the state of incipiently folding doubly spin-labeled protein within 50 microseconds of the start of folding. The distances and their distributions will be obtained from DQC and 17 GHz DEER measurements on the samples prepared using this freeze-quench apparatus. The feasibility of this technique is supported by recent distance measurements that we have completed for S47/K79 double mutants of iso-Cytochrome c in its folded state or unfolded by adding guanidium chloride (GdmCL) in several concentrations. In order to lengthen the nitroxide phase relaxation time Heme iron was reduced and the sample preparations were accomplished under anaerobic conditions. The measurements were performed using DEER set up for operation at 17.4 GHz. This working frequency let us use smaller samples and maintain high sensitivity. The GdmCl concentrations used were 0.7, 1.5, and 2.8M. In the folded state, the distance between spin labels was 15.8/0.5 [unreadable]. At [GdmCl] of 0.7 M the average distance was 30/3 [unreadable], whereas there was only a slight difference between the signals for 1.5M and 2.8M with average distances of 53/5 [unreadable] and 54/5 [unreadable] for the two cases, the same to within experimental error. Work is in progress to extract distance distributions from the dipolar signals by numerical methods based on Tikhonov regularization.